1. Field of the Invention
The invention relates to and has among its objects the provision of hybridomas that produce and secrete monoclonal antibodies which are specific for soybean Kunitz trypsin inhibitor, and to immunoassay methods for the determination of active Kunitz trypsin inhibitor in processed foods and soybean tissue utilizing the monoclonal antibodies.
2. Description of the Art
The protein of soybeans (Glycine max) is widely used in human foods in a variety of forms including infant formulas, tofu, soy protein isolates, soy flour, textured soy fibers, and soy sauce. Soybean protein products, properly processed, serve as an excellent source of low cost, high quality protein for human needs. Soybeans are also widely used as a component of animal feeds and as a major export commodity.
Protease inhibitors constitute at least 6% of the proteins of soybeans. There are two principal types of protease inhibitors found in soybeans, Kunitz trypsin inhibitor (KTI) and the double-headed Bowman-Birk trypsin and chymotrypsin inhibitors (BBI). It has been suggested that protease inhibitors in foods have both deleterious and beneficial effects. Animal studies suggest that active protease inhibitors may be toxic to humans, and may adversely affect nutritional quality; thus, there is an important need to minimize the amount of inhibitors in foods. Recent evidence suggests that some dietary protease inhibitors may have a beneficial anticarcinogenic effect. In addition, protease inhibitors may h=important in plant defenses against insects. Because inhibitors have both beneficial and adverse effects, a need exists to define the nature of inhibitors in foods and determine that is the best balance in the food to have optimum benefit.
Approaches to reduction of native (active) protease inhibitor content are Physical and chemical treatment of soy products and genetic alteration of the soybean crop. Although the protease inhibitor activity is largely inactivated by denaturation through conventionally applied heat treatments of soy flour, 10 to 15% residual activity remains. The problems with this approach are that the nature of the residual activity is difficult to characterize, and the process is costly in energy usage. Reduction of trypsin inhibitor content of soy products through genetic modification of the soybean plant to develop low Kunitz trypsin inhibitor has inherent limitations: desirable nutritional value and potential anticarcinogenic activity may be lost concomitant with the reduction of protease inhibitor content, cross pollination of the genetic variant by a wild-type cultivar could result in reexpression of protease inhibitor gene, and cultivars bred for low Kunitz trypsin inhibitor content would still contain other protease inhibitors. In addition, some protease inhibitors may function to prevent predation of the crops by insects, so that some protease inhibitory activities may be important for the agronomic value of soybean cultivars. With either genetic or physicochemical approaches to the reduction of protease inhibitor activity, the ability to measure low levels of protease inhibitors in soybean tissues and derived food products is essential. Since the effects of plant-derived protease inhibitors are of increasing importance in human and animal nutrition, food safety, and plant genetics, it is important to establish the exact protease inhibitor composition of a sample. What is needed is an assay which has the following characteristics: (1) it can differentiate between the two principal protease inhibitors, KTI and BBI, that is, it is specific for one or the other, (2) it is sufficiently sensitive and accurate to measure the low levels of inhibitors that are present in processed foods, and (3) it can differentiate between native (active) inhibitor and denatured inhibitor in processed samples.
Current assay techniques are not capable of providing this information. With regard to enzyme assays, because both KTI and BBI exist as several isoforms, which are derived from different genes or are produced by proteolysis, it is impossible to establish the exact protease inhibitor composition of a sample through enzymatic assay. Moreover, enzyme assays often give inaccurate results with processed samples having low residual activity as found in commercial foods.
Although immunochemical methods for estimating KTI were reported nearly two decades ago (N. Catsimpoolas and E. Leuthner, Analytical Biochemistry 31: 437-447 (1969) and N. Catsimpoolas et al., Cereal Chemistry 46: 136-144 (1969)), they have not been adopted by analytical or regulatory laboratories. These methods relied on precipitin analysis, which is insensitive, or binding of complement, an unstable mixture of serum proteins. These methods were applied to the detection of soybean trypsin inhibitor in raw meat products (Rossebo and Nordal, Z. Lebensmitt. Untersuch. Forsch. 148: 69-72 (1972)) and to the measurement of KTI in tissues of soybean plants (R. Freed and D. Ryan, Journal of Food Science 43: 1316-1319 (1978a) and Cereal Chemistry 55: 534-538 (1978b); A. Tan-Wilson et al., Plant Physiology 70: 493-497 (1982); and Hartl et al., Phytochemistry 25: 23-26 (1986)).
More recently, D. L. Brandon et al. (Abstract, 7th European Immunology Meeting, Jerusalem, Israel, Sept. 8-13, 1985) and D. Brandon and M. Friedman (Abstract, 6th International Congress of Immunology, Toronto, Canada, July 6-11, 1986) reported on polyclonal antibodies prepared by inoculation of rabbits with native KTI and KTI denatured by heat treatment or modified by reaction with agents which induce disulfide exchange. Polyclonal antibodies elicited with denatured KTI were specific only for denatured KTI. In contrast, polyclonal antibodies elicited by native KTI recognized both native and heat-treated KTI indicating that antibodies specific to native KTI could not be produced in rabbits using conventional protcols. Brandon and Friedman, 1986, supra, also reported that preliminary results indicated that monoclonal antibodies to KTI could be produced and that three epitopes (antiboding sites) were identified as present on the molecule. One of these epitopes was retained when pure KTI was denatured. The conditions used were those to prepare a sample for gel electrophoresis -- treatment in solution containing detergent and reducing agent (ca. 1% sodium dodecylsulfate and 0.1% 2-mercaptoethanol) at 100.degree. C. for 2 minutes (Brandon and Friedman, unpublished information). These conditions are known to unfold proteins completely, destroying disulfide bonds and other features of the secondary and tertiary structure of proteins and are unlike the conditions used in food processing (moist heat or alkaline treatment in the absence of detergent and reducing agent).
The production of monoclonal antibodies by fusion of spleen cells and myeloma cells has been described previously by Kohler and Milstein, Nature 256: 495-497 (1975) and many other investigators. While monoclonal antibodies against protease inhibitors from animal sources are known (See Herion et al., Bioscience Reports 4: 139-148 (1984)), no immunoassays have been described which use monoclonal antibodies to detect specific protease inhibitors in complex samples such as soybeans and processed foods. It was unknown whether sufficiently specific antibodies could be prepared which could differentiate between KTI and BBI, distinguish between native KTI and KTI denatured by heat, and would be useful in detecting low levels of KTI in samples containing multiple protease inhibitors and containing phytate, fat, fiber, and other potentially interfering substances. Further, it was uncertain whether antibodies and assay methods could be developed to identify the particular KTI isoforms present in a sample and to identify whether KTI was bound to trypsin or trypsin-like enzymes. These specificities would be useful in analyzing samples of unknown origin, or for identifying which plant cultivars are represented in a sample. The identification of KTI as part of a complex would allow quantitation under conditions where enzymatic assays would be inaccurate or impossible. Quantitation of complexes would also be important in studying allergic manifestations of soy-containing diets.
While various immunoassays, including enzyme-linked immunosorbent assay (ELISA) methods have been used for quantitation of plasma protease inhibitors, it has not been previously demonstrated that protease inhibitors in general, or KTI specifically, can be labeled with an enzyme in such a way that the antigenic structure is retained. Retention of antigenic structure is essential to achieve the specificity required of the immunoassays. Further, it is known that some proteins undergo structural changes which affect their antigenicity when adsorbed onto plastic assay plates (Dierks at al., Molecular Immunology 23: 403-411 (1986)), so it could not be predicted that the antigenic differences among KTI isoforms and their trypsin complexes would be readily measurable utilizing solid-phase assay formats.